2 replies to this topic

[vinayadass]

I am trying to measure IgG1 from purified mouse B cell culture supernatant by ELISA. I have an rat anti-mouse Ab which I am testing for its B cell activating abilities in general and in vitro Ig class switching in particular.  When I add this Ab (over a wide range of dilutions) to B cell cultures with or without LPS or CD40 or LPS/IL4, the color development is very strong. I suspected that my Ab (purified via ascitis over protein G column) may have traces of mouse Ig (via blood). However, when adsorbed my Ab with anti-mouse IgG-agarose beads, the result was still the same. I am now guessing that my secondary Ab (goat anti-mouse IgG1-HRP from Southern Biotech) may be cross reacting with the the Rat IgG (from added Ab) in the culture supernatant and could be the culprit. Here is where I need your help. I have looked up Biocompare.com without luck to see if there are any vendors who have rat adsorbed anti-mouse IgG1-HRP. I shall appreciate if any of you could help me locating such vendor.

Thank you.

[Prep!]

vinayadass, on Mar 24 2010, 05:05 AM, said:

I am trying to measure IgG1 from purified mouse B cell culture supernatant by ELISA. I have an rat anti-mouse Ab which I am testing for its B cell activating abilities in general and in vitro Ig class switching in particular.  When I add this Ab (over a wide range of dilutions) to B cell cultures with or without LPS or CD40 or LPS/IL4, the color development is very strong. I suspected that my Ab (purified via ascitis over protein G column) may have traces of mouse Ig (via blood). However, when adsorbed my Ab with anti-mouse IgG-agarose beads, the result was still the same. I am now guessing that my secondary Ab (goat anti-mouse IgG1-HRP from Southern Biotech) may be cross reacting with the the Rat IgG (from added Ab) in the culture supernatant and could be the culprit. Here is where I need your help. I have looked up Biocompare.com without luck to see if there are any vendors who have rat adsorbed anti-mouse IgG1-HRP. I shall appreciate if any of you could help me locating such vendor.

Thank you.

Are u talking abt this??

http://products.invi.../product/046120

[vinayadass]

Pradeep Iyer, on Mar 23 2010, 08:24 PM, said:

vinayadass, on Mar 24 2010, 05:05 AM, said:

I am trying to measure IgG1 from purified mouse B cell culture supernatant by ELISA. I have an rat anti-mouse Ab which I am testing for its B cell activating abilities in general and in vitro Ig class switching in particular.  When I add this Ab (over a wide range of dilutions) to B cell cultures with or without LPS or CD40 or LPS/IL4, the color development is very strong. I suspected that my Ab (purified via ascitis over protein G column) may have traces of mouse Ig (via blood). However, when adsorbed my Ab with anti-mouse IgG-agarose beads, the result was still the same. I am now guessing that my secondary Ab (goat anti-mouse IgG1-HRP from Southern Biotech) may be cross reacting with the the Rat IgG (from added Ab) in the culture supernatant and could be the culprit. Here is where I need your help. I have looked up Biocompare.com without luck to see if there are any vendors who have rat adsorbed anti-mouse IgG1-HRP. I shall appreciate if any of you could help me locating such vendor.

Thank you.

Are u talking abt this??

http://products.invi.../product/046120

........
The Ab apparently has been pre-adsorbed to remove mouse non-IgG1, swine, and human cross reactivity but doesn't say whether rat IgG cross reactivity has been taken care of. I do not think it will serve my purpose.

Thank you for the help.